CN112076179A

CN112076179A - Medical application of honokiol

Info

Publication number: CN112076179A
Application number: CN202011033093.XA
Authority: CN
Inventors: 李文斌; 李生兰; 邱能; 叶昊宇
Original assignee: Chengdu Jinrui Foundation Biotech Co ltd; Beijing Tiantan Hospital
Current assignee: Chengdu Jinrui Foundation Biotech Co ltd; Beijing Tiantan Hospital
Priority date: 2020-09-27
Filing date: 2020-09-27
Publication date: 2020-12-15
Also published as: WO2022062197A1; CN115252589A

Abstract

The invention relates to the medical application of honokiol, in particular to the application of honokiol in inhibiting medulloblastoma, and experiments prove that the honokiol can inhibit the cell proliferation of medulloblastoma, induce the cell cycle retardation of medulloblastoma and induce the cell apoptosis of medulloblastoma; the application of honokiol in promoting hair growth is proved by experiments, and the honokiol can promote hair growth and has no toxic or side effect on liver and kidney; and the application of the honokiol in promoting the blackening of the white hair, and experiments prove that the honokiol can promote the blackening of the white hair and has no toxic or side effect on liver and kidney.

Description

Medical application of honokiol

Technical Field

The invention relates to the field of medicines, in particular to medical application of honokiol. More specifically, the present invention relates to the use of honokiol in the inhibition of medulloblastoma, and to the use of honokiol for hair growth and for promoting the darkening of white hair.

Background

Honokiol, known as Honokiol in english, has a chemical name of 3', 5-di-2-propenyl-1, 1' -biphenyl-2, 4' -diol, and has the following structural formula:

honokiol is a small molecular compound with wide biological activity extracted and separated from cortex of Magnolia officinalis (Magnolia officinalis rehd. et Wils.), and the main biological activity of honokiol comprises inflammation resistance, microorganism resistance, ulcer resistance, oxidation resistance, anxiety resistance, depression resistance, thrombus resistance, aging resistance, cholesterol reduction and the like.

In view of the wide medicinal value of honokiol, further research on new application of honokiol is needed.

Disclosure of Invention

Through a large number of experimental researches, the inventor discovers a new application of honokiol, and the honokiol can be used for inhibiting medulloblastoma. It is therefore an object of the present invention to provide the use of honokiol in the manufacture of a medicament for the inhibition of medulloblastoma.

The use according to the present invention for inhibiting medulloblastoma, wherein the honokiol is prepared as honokiol liposomes (Lip-HNK or Lip-HK), preferably as injectable honokiol liposomes.

The use according to the invention for inhibiting medulloblastoma, wherein the honokiol inhibits cell proliferation of medulloblastoma.

The use according to the invention for inhibiting medulloblastoma, wherein the honokiol induces apoptosis of the medulloblastoma. In one aspect, the honokiol induces apoptosis of medulloblastoma cells via the ROS/ERK/p38MAPK pathway, wherein the honokiol-induced apoptosis of medulloblastoma cells involves ROS generation, and inhibits the ERK/p38MAPK signaling pathway by generating excess ROS within medulloblastoma cells. On the other hand, the honokiol induces apoptosis of medulloblastoma cells through a Caspase (cysteine-containing aspartate proteolytic enzyme) dependent pathway.

The use according to the invention for inhibiting medulloblastoma, wherein the honokiol induces cell cycle arrest of medulloblastoma. Specifically, the honokiol induces cell G1 cycle arrest in medulloblastoma.

The invention researches the inhibition effect and mechanism of the Lip-HNK on the proliferation of medulloblastoma cells. The Lip-HNK can also induce cycle arrest of medulloblastoma cells G1 and caspase-dependent apoptosis, but has no obvious cytotoxicity to normal cells. Lip-HNK has been shown to inhibit the growth of tumor cell lines, but the use of Lip-HNK for inhibiting medulloblastoma has not been reported, and the molecular mechanism of the effect of Lip-HNK on medulloblastoma cell death has not been studied. This inhibition of medulloblastoma by Lip-HNK may be mediated by the induction of intracellular Reactive Oxygen Species (ROS) and mitochondrial membrane potential loss. Meanwhile, the Lip-HNK has a dose-dependent inhibition effect on the phosphorylation of ERK and p 38. More importantly, the effect of Lip-HNK on mitochondrial membrane potential, ROS generation and phosphorylation of ERK, p38 was found to be significantly reversed by ROS inhibitor, suggesting that Lip-HNK affects medulloblastoma cells and ERK/p38MAPK signaling by generating excessive amounts of ROS. Therefore, the inventor of the invention firstly clarifies that the medulloblastoma induces the apoptosis of the medulloblastoma through the ROS/ERK/p38MAPK pathway, and provides a basic scientific basis for the new treatment potential of the Lip-HNK for treating the medulloblastoma.

In addition, through a large number of experimental studies, the inventors also found that honokiol can be used to promote hair growth. It is therefore a further object of the present invention to provide the use of honokiol in the manufacture of a medicament for promoting hair growth.

The use according to the present invention for promoting hair growth, wherein the honokiol is prepared as honokiol liposomes, preferably injectable honokiol liposomes.

The use according to the present invention for promoting hair growth, wherein the site of hair growth may be the head.

The invention researches the influence of honokiol on hair growth, and experiments prove that the honokiol can promote the hair growth, particularly, the honokiol can accelerate the hair growth speed and increase the hair follicle length, and has no toxic or side effect on liver and kidney.

In addition, through a great deal of experimental research, the inventor also finds that honokiol can be used for promoting the blackening of white hair. It is therefore a further object of the present invention to provide the use of honokiol in the manufacture of a medicament for promoting the darkening of white hair.

The use according to the present invention for promoting blackening of white hair, wherein the honokiol is prepared as a honokiol liposome, preferably an injectable honokiol liposome.

The invention researches the influence of honokiol on blackening of white hair, and experiments prove that the honokiol can promote the blackening of the white hair and has no toxic or side effect on liver and kidney.

It is still another object of the present invention to provide honokiol liposomes for inhibiting medulloblastoma, promoting hair growth, and promoting blackening of white hair.

The honokiol liposome according to the invention is an injection honokiol liposome.

The honokiol liposome according to the invention, wherein the honokiol liposome can be in the following dosage forms: freeze-dried powder preparations, including injection freeze-dried powder preparations and oral freeze-dried powder preparations; tablets, including immediate release tablets and sustained release tablets; capsules, including hard capsules, soft capsules, sustained release capsules, and enteric capsules; transdermal formulations, and the like.

The honokiol liposomes according to the invention, wherein the honokiol liposomes can be administered by the following routes: intravenous injection, intramuscular injection, subcutaneous injection, oral administration, ocular administration, pulmonary administration, transdermal administration, nasal administration, and the like.

Drawings

Fig. 1A-1C show that Lip-HNK inhibits cell proliferation of medulloblastoma.

Fig. 2A-2E show that Lip-HNK induces cell cycle arrest in medulloblastoma.

Figures 3A-3D show that Lip-HNK induces apoptotic cell death in medulloblastoma.

Figures 4A-4F show that Lip-HNK-induced apoptosis of medulloblastoma cells is involved in ROS generation.

Fig. 5A-5C show that Lip-HNK inhibits the ERK/p38MAPK signaling pathway by generating excess ROS in medulloblastoma cells.

FIGS. 6A-6C show that Lip-HNK induces apoptotic cell death in medulloblastoma cells via a Caspase-dependent pathway.

Fig. 7 shows hair growth at different times in the saline group and the Lip-HNK group.

Fig. 8 shows HE sections of hair follicles of skin tissue on the back of mice at different treatment time points.

Figure 9 shows that Lip-HNK has no toxic side effects on the liver and kidney of mice.

Fig. 10 shows that honokiol promotes hair growth and blackening of white hair.

Detailed description of the preferred embodiments

The following experimental examples are provided to further illustrate the medical use of honokiol.

Experimental example 1: use of honokiol for inhibiting medulloblastoma

1. Experimental materials and instruments:

the honokiol liposome is from Chengdu Jinrui industries Biotech Co., Ltd; human medulloblastoma cells (DAOY cells and D283 cells) mouse glial BV2 cells and mouse hippocampal neuronal HT22 cells were purchased from cell banks in the foundation of the beijing consortium.

PBS phosphate buffer, 4% paraformaldehyde, 0.5% crystal violet, methanol, absolute ethanol, CCK-8, active oxygen kit, fetal bovine serum (Gbico), and antibody: caspase-3 (cat # ab13847), c-caspase-3(ab32042), ERK (ab17942), p-ERK (ab201015), p38(ab170099), secondary antibodies all available from Abcam corporation; p-p38(8690T), CDK4(12790) available from Cell Signaling Technology; GAPDH (China fir gold bridge), Hoechst33342, propidium iodide PI, apoptosis kit were purchased from BD company; JC-10 kit (CA310-100, Solibao, Inc.), RIPA lysate (R0020, Solibao), PVDF membrane (ISEQ00010 Solibao), 96-well plate, six-well plate, and petri dish were purchased from Corning, Inc.; fluorescence microscope, enzyme labeling instrument, flow cytometry (BD Biosciences, San Jose, CA, USA), electrophoresis apparatus, electrotransformation apparatus, cell culture box, all from Beijing Tiantan hospital foundation and transformation research laboratory.

2. Experimental methods and results

The following cell experiments were performed to analyze the effect and mechanism of honokiol liposomes on medulloblastoma, respectively.

Cell viability assay

Cell viability was determined by the CCK-8 method, 2X 10 for DAOY and D283 cells³The cells/well speed were seeded in 96-well plates for 24 hours and then treated with different concentrations of Lip HNK for 48 h. Before the end of the treatment, 10. mu.L of CCK-8 solution was added to each well. After 1 hour of incubation, the absorbance was measured at 450 nm.

Clone formation assay

In the clone formation assay, DAOY cells were cultured at 1X 10³Density of individual cells/well were placed in 6-well plates and incubated with different concentrations of Lip HNK at 37 ℃. Fresh medium was then changed daily and cultured for 14 days. After fixation with 4% paraformaldehyde and staining with 0.5% crystal violet for 15min, the number of clones was observed.

Fig. 1A-1C show that Lip-HNK inhibits cell proliferation of medulloblastoma, wherein fig. 1A shows cell viability graphs of medulloblastoma cells (DAOY, D283) and normal cells (BV2, HT22) treated with different concentrations of Lip-HNK (0, 20,30,40,50 μ M); FIG. 1B shows the cell morphology changes induced by Lip-HNK treatment; fig. 1C shows representative images of a colony formation assay for DAOY cells.

Cell cycle analysis DAOY and D283 cells at 5X 10⁵Individual cells/well density were seeded onto 6-well plates and treated with different concentrations of Lip HNK for 48 hours. Collecting floating and adherent cells, cells fixed in 70% ethanolAt least 24 hours at minus 20 degrees. After all cells were fixed, periodic assays were performed. The method comprises the following specific steps: after fixation, the cells were added to 5 ml of cold PBS and centrifuged at 1500rpm for 10 minutes. The supernatant was removed, leaving a pellet of cells. The cell pellet was resuspended in 2 ml of cold PBS and centrifuged at 1500rpm for 10 minutes to obtain a cell pellet. Finally, the cell pellet was resuspended in 2 ml of cold 2% FBS/PBS and centrifuged at 1500rpm for 10 minutes to obtain a cell pellet. The cells were resuspended in the appropriate amount of PI/RNAase stain, and stained at room temperature in the dark for 30 minutes before testing on the machine.

Hoechst33342 staining

Hoechst33342 staining, DAOY cells and D283 cells were pretreated with different concentrations of Lip-HNK (0, 20,30, 40. mu.M) for 48h, washed with cold PBS, fixed with cold methanol, and stained with Hoechst33342 (1. mu.g/mL) for 15min, and morphological characteristics of apoptotic cells were observed with a fluorescence microscope.

Detection of apoptosis by Annexin V and PI staining method

Apoptotic cell death was determined using the apoptosis detection kit (Annexin V-PI: BD Biosciences, San Jose, CA, USA). Treatment of 5X 10 with different concentrations of Lip-HNK (0, 20,30, 40. mu.M)⁵DAOY cells and D283 cells for 48h, adherent cells and detached cells were taken, washed once with PBS, stained with Annexin V-FITC and Propidium Iodide (PI) at 37 ℃ for 15min, and apoptosis was detected by flow cytometry.

Fig. 2A-2E show that Lip-HNK induces cell cycle arrest in medulloblastoma, where fig. 2A and 2C show the cell cycle distribution of DAOY cells and D283 cells treated with different concentrations of Lip-HNK; fig. 2B and 2D show representative images of cell cycle distribution (%) of DAOY cells and D283 cells analyzed by flow cytometry; FIG. 2E shows P21 protein expression levels examined by western blot 48 hours after treatment of DAOY and D283 cells with Lip-HNK.

Fig. 3A-3D show that Lip-HNK induces apoptotic cell death of medulloblastoma, wherein fig. 3A shows analysis of nuclear structure of DAOY cells by fluorescence microscopy; FIG. 3B shows the ratio of PI staining (red) to Hoechst33342 staining (blue), representing cell mortality; FIG. 3C shows determination of apoptotic cell death by Annexin V/PI flow cytometry analysis; figure 3D shows the percentage of apoptotic cells.

Intracellular ROS detection

DAOY cells and D283 cells were treated with different concentrations of Lip-HNK (0, 20,30, 40. mu.M) for 48h, then washed with cold PBS and incubated in 10. mu.M DCFH-DA at 37 ℃ for 30 min in the dark. DCF fluorescence was measured using a flow cytometer (BD Biosciences, San Jose, CA, USA) and data was analyzed using FlowJo 10. Fluorescence intensity of intracellular DCF represents ROS levels and Image J was used to quantify fluorescence intensity.

Figures 4A-4F show Lip-HNK-induced apoptosis of medulloblastoma cells involving ROS generation, wherein figure 4A shows ROS generation in Lip-HNK treated cells as measured by fluorescence microscopy using DCFH-DA staining; FIG. 4B shows the relative DCF fluorescence intensity in three groups (control, Lip-HNK and Lip-HNK + NAC (N-acetyl-L-cysteine)) expressed as a multiple of the fluorescence intensity of the Lip-HNK and Lip-HNK + NAC groups relative to the control group; figure 4C shows measurement of NAC inhibition of honokiol liposome-induced ROS generation by flow cytometry; FIG. 4D shows the percent cell viability as determined by CCK-8; FIGS. 4E and 4F show the staining of cells with Annexin V/PI by flow cytometry analysis.

FIGS. 5A-5C show that Lip-HNK inhibits the ERK/p38MAPK signaling pathway by generating excess ROS in medulloblastoma cells, wherein FIG. 5A shows the protein levels of p-ERK, p-p38, p38 in DAOY cells and D283 cells after Lip-HNK treatment; FIG. 5B shows that Lip-HNK acts on medulloblastoma cells by ERK/p38MAPK phosphorylation, and the levels of p-ERK, p-p38, p38 protein were detected by immunoblotting after co-treating medulloblastoma cells with 40 μ M Lip-HNK and 5mM NAC; FIG. 5C shows detection of protein levels of apoptosis-related factors (including cleaved Caspase3, Caspase3, Bax and Bcl-2) by immunoblotting.

Mitochondrial membrane potential determination

Mitochondrial membrane potential was measured using JC-10 kit. DAOY cells and D283 cells (2X 10)⁵) Inoculation, treatment with Lip-HNK for 48h, followed by incubation with JC-10 for 30 min at 37 ℃ and two washes with PBS. Flow cytometry detection of MMP (mitochondrial membrane potential) changes. The positive control was treated with CCCP (active oxygen positive control reagent).

FIGS. 6A-6C show that Lip-HNK induces apoptotic cell death in medulloblastoma cells via a Caspase-dependent pathway, wherein FIG. 6A shows evaluation of expression levels of apoptotic proteins (Bcl-2, Bax, Caspase-3 and lytic Caspase-3) by Western blot; figure 6B shows data obtained from at least three independent experiments, wherein the values are mean ± SD, p <0.05, p < 0.01; figure 6C shows MMP evaluation using a fluorescent mitochondrial probe JC-10, where red/green fluorescence intensity was analyzed by flow cytometry.

The experimental results show that Lip-HNK has an inhibitory effect on medulloblastoma cells. In one aspect, Lip-HNK induces apoptosis of medulloblastoma cells via the ROS/ERK/p38MAPK pathway; on the other hand, Lip-HNK can also induce cycle arrest of medulloblastoma cells G1 and caspase-dependent apoptosis, but has no obvious cytotoxicity to normal cells. The inhibitory effect of Lip-HNK on medulloblastoma is mediated by the induction of intracellular ROS and mitochondrial membrane potential loss. Meanwhile, the Lip-HNK has a dose-dependent inhibition effect on the phosphorylation of ERK and p 38. The influence of the Lip-HNK on mitochondrial membrane potential, ROS generation and ERK and p38 phosphorylation can be remarkably reversed by the ROS inhibitor, and the fact that the Lip-HNK influences medulloblastoma cells and ERK/p38MAPK signaling through excessive ROS generation is shown to provide a basic scientific basis for the new treatment potential of the Lip-HNK for treating medulloblastoma.

Experimental example 2: use of honokiol for promoting hair growth

1. Experimental materials and instruments

The honokiol liposome is from Chengdu Jinrui Bio-technology Co., Ltd, and the C57BL/6 mouse is purchased from Beijing Wintotonglihua laboratory animal technology Co., Ltd; 4% paraformaldehyde (manufacturer: Biyuntian; cat # P0099), 5% chloral hydrate (Shanghai Yuanmu R18184), depilatory cream, syringe, PBS, distilled/tap water, xylene, gradient ethanol, neutral gum, paraffin embedding machine, slicer, baking machine, microscope all from Beijing Tiantan hospital foundation and transformation research laboratory.

2. Experimental methods and results

A6-week-old C57BL/6 mouse with a weight of 18-20g was used as an experimental animal, and the animal was allowed to acclimatize for three days after purchase, followed by a back depilatory treatment, and an animal experiment was carried out at a concentration of 20mg/kg of honokiol liposomes. The HE staining technology and SPSS software are adopted to statistically compare the hair growth conditions of mice of different treatment groups on different administration days (10 days, 14 days and 21 days), including the days of skin color change in an administration area, the days of starting new hair growth, the length of hair follicles and the like, so as to analyze the influence of honokiol liposome on the hair regeneration of the mice. The specific experiment is as follows:

1) hair growth in mice at different times

Weighing the weight of a 6-week-old C57BL/6 mouse, then giving 5% chloral hydrate for anesthesia, then giving hair removal on the back by using a hair removal paste, finishing photographing after hair removal, performing intraperitoneal injection of physiological saline and honokiol liposome (20mg/kg) every day after the next day after hair removal, observing the growth condition of skin and hair every day, performing hair measurement after hair growth, photographing respectively at 0, 10, 14 and 21 days after hair removal, taking the skin and liver and kidney tissues for paraffin embedding, and simultaneously reserving the tissues for storage in liquid nitrogen. Fig. 7 and tables 1 and 2 show that the skin darkening time and hair growth time of the honokiol liposome group were shorter than those of the saline control group.

TABLE 1 time to dark skin color in the mouse administration area after administration

TABLE 2 time to onset of growth of new hair in mice after dosing

2) HE section of skin tissue hair follicle on back of mouse at different treatment time points

The skin tissues at

day

0, 10, 14, 21 after depilation were paraffin-embedded and HE stained.

Paraffin embedding: taking fresh skin tissue, and cutting into tissue blocks of 3-5mm × 3-5mm × 10-20mm size with a blade;

fixing: and (3) placing the cut tissue blocks into 4% paraformaldehyde for fixation, wherein the volume ratio of the tissue blocks to the 4% paraformaldehyde is 1: preferably 20; after fixation, washing with PBS for 3 times, 5 minutes each time;

and (3) dehydrating and transparency: the step should set proper time according to different organizations, and the basic flow is as follows: 75% ethanol-85% ethanol-95% ethanol 1-95% ethanol 2-absolute ethanol 1-absolute ethanol 2-xylene 1-xylene 2-xylene 3;

wax dipping: melting paraffin, and keeping the temperature at about 57 ℃;

embedding: the tissue block is placed in a mould containing wax liquid, the required tissue section is parallel to the bottom, and the wax liquid is easy to solidify in a cold environment, so that the step is as fast as possible.

The HE staining method comprises (1) immersing the slices in xylene for 5-10 min; (2) immersing slices in xylene for 5-10 min; (3) 100% alcohol for 1 min; (4) 100% alcohol for 1 min; (5) 95% ethanol for 1 min; (6) 95% ethanol for 1 min; (7) 90% alcohol for 1 min; (8) 80% ethanol for 1 min; (9) washing with tap water for 1 min; (10) immersing into hematoxylin staining solution for 10-15 min; (11) washing with tap water for 30sec-1 min; (12) differentiating with 1% hydrochloric acid alcohol for 30 sec; (13) flushing with running water for more than 15 min; (14) staining with 1% eosin alcohol for 3-5 min; (15) differentiation with 90% or 95% alcohol for 30 sec; (16) 95% alcohol for 30sec-1 min; (17) 95% alcohol for 30sec-1 min; (18) 95% alcohol for 30sec-1 min; (19) 100% alcohol for 1 min; (20) 100% alcohol for 1-2 min; (21) 1min of dimethylbenzene carbonate; (22) xylene for 1-2 min; (23) xylene for 1-2 min; (24) xylene for 1-2 min; (25) and (5) sealing by using neutral gum.

Fig. 8 shows that the hair follicle growth cycle of the honokiol liposome group was earlier than that of the saline control group, and the number of hair follicles was significantly increased than that of the saline control group. Also, table 3 shows that the length of the hair follicle in the honokiol liposome group is greater than that in the saline control group.

TABLE 3 analysis of hair follicle length in mice at different treatment times

3) Hepatotoxicity and hepatotoxicity

In order to further determine whether the honokiol liposome has toxic reaction while promoting hair growth, the liver and kidney tissues are embedded in paraffin and then subjected to HE staining (the HE staining operation of the liver and kidney tissues is the same as that of the skin tissues). Fig. 9 shows that there was no significant hepatorenal toxicity after intraperitoneal injection of honokiol liposomes, and mice did not die and had no effect on body weight, compared to the normal saline control group.

In addition, in clinical studies of honokiol liposomes for the treatment of brain gliomas, the inventors found that honokiol can promote hair growth and blacken white hair. Figure 10 shows that the patient's hair growth increased and white hair became black after intravenous injection of honokiol liposomes.

Claims

1. Use of honokiol in the preparation of a medicament for inhibiting medulloblastoma.

2. The use according to claim 1, wherein the honokiol is prepared as honokiol liposomes, preferably injectable honokiol liposomes.

3. The use of claim 1, wherein the honokiol inhibits cell proliferation of medulloblastoma.

4. The use of claim 1, wherein the honokiol induces apoptosis of medulloblastoma.

5. The use of claim 4, wherein the honokiol induces apoptosis of medulloblastoma cells via the ROS/ERK/p38MAPK pathway.

6. The use of claim 4, wherein the honokiol induces apoptosis of medulloblastoma cells via a Caspase-dependent pathway.

7. The use according to claim 1, wherein the honokiol induces cell cycle arrest of medulloblastoma, preferably the honokiol induces cell G1 cycle arrest of medulloblastoma.

8. Use of honokiol in the manufacture of a medicament for promoting hair growth.

9. Use according to claim 8, wherein the site of hair growth is the head.

10. Use of honokiol in the preparation of a medicament for promoting the blackening of white hair.